This program project brings together six investigators from four institutions to work on three major problems in clinical medicine, namely the treatment of pain, tolerance and dependence, and myocardial ischemia. During the past 4 years, we have made significant progress towards the development of an opioid analgesic ([Dmt1]DALDA) that is superior compared to morphine in terms of potency, duration of action, and side effect profile. [Dmt1]DALDA has extremely high potency after intrathecal and systemic administration, is very long-acting, has no significant adverse effects, and can even protect cardiac function against ischemia-reperfusion injury. Furthermore, morphine-tolerant animals showed no cross-tolerance to [Dmt1]DALDA. The systemic potency of this highly polar 3+ net charge peptide was unexpected, and the ability of [Dmt1]DALDA to penetrate the BBB was a surprise. Our results show that [Dmt1]DALDA has enormous potential as a therapeutic agent. Its success was unexpected and led us to question our prevailing knowledge and understanding in opioid pharmacology and peptide pharmacokinetics. A number of novel hypotheses 1 were prompted by the findings with [Dmt1]DALDA and will be tested in this competitive renewal application. The overall specific aims of this Program Project are to determine: (i) the structural requirements for small peptides to penetrate cell membranes; (ii) the role of transporters and membrane permeability in peptide pharmacokinetics; (iii) the mechanisms behind the extraordinary antinociceptive potency of [Dmt1]DALDA, (iv) the reason why there is no cross-tolerance to [Dmt1]DALDA in morphine-tolerant animals; (v) the reason why tolerance to [Dmt1]DALDA is limited to the spinal cord; (vi) the ability of [Dmt1]DALDA to protect the heart during prolonged ischemia and reperfusion. The specific aims will be accomplished with the development of novel [Dmt1]DALDA analogs that will permit structure-activity relationships in animal and cellular studies, and radiolabelled and fluorescent analogs for cellular and biochemical studies. The information to be gained from the proposed studies may significantly influence the approach to peptide drug design and the treatment of pain, addiction, heart attacks and strokes. [unreadable] [unreadable]